Expression Of Three 2,3-Dihydroxybiphenyl 1,2-Dioxygenase Genes From Biphenyl-Degrading Strain BP3 And Characterization Of The Products

Polychlorinated biphenyls (PCBs) are important raw materials, which are widely used in the industry. Many researches reveal that PCBs are carcinogenic to human. The study on microbial degradation of PCBs is one of the hot topics in the scope of environmental microbiology. Aerobic microbial degradation of PCBs is via the biphenyl metabolic pathway; the ring cleavage of 2,3-DHBP is an important reaction in this pathway, which is catalyzed by 2,3-dihydroxybiphenyl 1,2-dioxygenase.In our previous study, a biphenyl degrading bacteria Achromobacter sp. BP3 was isolated. Its genomic library was constructed and screened, three positive colonies harboring the activity of 2,3-dihydroxybiphenyl 1,2-dioxygenase were obtained, their recombinant plasmids were p118-2, p118-3 and p118-5, the inserted DNA fragments were sequenced and analyzed respectively; three 2,3-dihydroxybiphenyl 1,2-dioxygenase encoding genes bphC, bphC2 and xylE were deduced.In the present study, these three genes were cloned to the expressing vector pET29a and functionally expressed in E. coli BL21 (DE3) as a C-terminal 6-His tagged fusion protein, respectively. The expressed product was purified with Ni-Agarose gel affinity column and subjected to the enzymatic properties study.The investigation on the enzymatic properties showed that the optimal catalyzing temperature for BphC, BphC2 and XylE was 30℃,30℃and 50℃, respectively; the optimal reaction pH was 8.0,9.0 and 9.0, respectively; on the concentration of 1 mmol/L of metal ions, Cu2+ and Co2+ showed inhibitory effect on all of them, but showed the strongest influence against BphC(the rate is above 90%); Mg2+ showed no evident influence on BphC, while showed inhibition against BphC2 and XylE; Fe2+ could promote the activity of BphC2 and XylE, indicting that they were Fe2+-dependent dioxygenases; BphC and BphC2 had higher substrate affinity on the bicyclic substrate 2,3-DHBP, while XylE exhibited the higher substrate affinity on the simple ring substrate. The expression of bphC, bphC2 and xylE in the strain BP3 was also investigated. The results showed that bphC was constitutively expressed, bphC2 was induced by biphenyl, and xylE was induced by both biphenyl and toluene.